Genomic instability is a common feature of many cancers. Indeed, oncogene expression is sufficient to cause replication stress. Replication stress is defined as inefficient DNA replication associated with DNA replication fork slowing or stalling, accompanied by helicase-polymerase uncoupling. This perturbation in DNA replication leads to the activation of a DNA damage response (DDR), regulated by the ATR and ATM kinases, which stabilize stalled forks and promote double strand break (DSB) processing, respectively. Engagement of the DDR acts as a barrier against tumorigenesis by inducing cell cycle arrest, senescence, or apoptosis. Determining how oncogenes cause replication stress has been a focus of recent research. Multiple models have been proposed, including oxidative damage, nucleotide deficiency, and premature origin firing~ however, much remains to be understood. The goal of my proposal is to increase our understanding of how oncogenic stress causes genomic instability. In this research I will utilize two distinct approaches: one that examines how alterations in cytoplasmic signaling events promote oncogene-induced DDR and another that determines the specific effects of oncogene expression on replisome composition, which results in an increased reliance on ATR to minimize fork collapse into DNA double strand breaks. For the first objective, I will determine if the intracellula signaling perturbations, specifically, unequal amplitude between parallel pathways, is the original source of the mechanism that ultimately leads to DDR activation in oncogene-expressing cells. To test this model, I have generated cell-based systems to induce such a signaling imbalance between the two main pathways activated by growth factor receptors, namely the MAPK and PI3K pathways. These systems will be used to examine if greater activation of one pathway over the other leads to increased replication stress and DDR. In addition, I will distinguish these effects from an increase in signaling amplitude rom both pathways. For my second objective, I will use DNA combing to investigate if oncogene expression affects replication fork dynamics in a manner that requires ATR signaling for continued processivity. These experiments will determine if oncogenic stress slows DNA replication in a manner that results in helicase-polymerase uncoupling, the key means by which ATR is activated at the replication fork. Lastly, I will determine if replication stress is associated with a premature disassociation of replication factors rom the fork. Several techniques will be used to detect changes in the representation of replisome components under distinct oncogenic stresses, namely Aurora A (AURKA) and MYC overexpression as well as oncogenic KRASG12V expression. Together, these experiments will further define how altered signal transduction under oncogenic stress alters replication fork dynamics and promotes the DDR.